Endothelial cell (EC) phenotypes display remarkable heterogeneity in health and disease. An important goal in vascular biology is to understand the molecular mechanisms underlying the spatial and temporal modulation of EC phenotypes. This competing renewal application is an extension of our previous work aimed towards elucidating the role of forkhead (FOXO) transcription factors in EC biology. Three novel and exciting aspects of FOXO biology have emerged in the past 3 years, which together with our own published data and preliminary results provide a strong foundation for the current proposal: 1) despite the fact that FOXO proteins are ubiquitously expressed, FOXO- deficient mice display a vascular phenotype, whether the gene is deleted during embryogenesis or in adults, 2) the vascular phenotype is organ-specific and FOXO-deficient ECs from different organs demonstrate different gene expression patterns and functional changes, and 3) FOXO proteins (at least in other cell types) have been shown to bind to and/or interact with a broad spectrum of transcription factors. Based on these considerations, we hypothesize that FOXO proteins (in particular FOXO1) cooperate with other transcription factors to regulate EC phenotypes. The overall goal is to identify FOXO1-associated transcriptional networks in ECs, to understand how these mediate vascular bed-specific gene expression, and to define the in vivo role of FOXO1 in the intact endothelium. Aim 1 is to characterize functional interactions between FOXO1 and other transcription factors in ECs. Aim 2 is to characterize combinatorial gene regulation by FOXO1 and NF-kB in ECs. Aim 3 is to analyze the effect of conditional FOXO1 over-expression and knockout on EC function and dysfunction in normal and pathological states. PUBLIC HEALTH RELEVANCE: The proposal is significant in that it will provide important insights into the mechanisms (hence therapeutic potential) of FOXO1-mediated EC signal transduction.